İSTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY REGULATION OF MICROTUBULE SEVERING M.Sc. Thesis by Şirin KORULU, B.Sc. Department: Advanced Technologies in Engineering Programme: Molecular Biology–Genetics and Biotechnology Supervisor: Assoc. Prof. Dr. Arzu KARABAY KORKMAZ JUNE 2006
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İSTANBUL TECHNICAL UNIVERSITY ���� INSTITUTE OF SCIENCE AND TECHNOLOGY
REGULATION OF MICROTUBULE SEVERING
M.Sc. Thesis by
Şirin KORULU, B.Sc.
Department: Advanced Technologies in Engineering
Programme: Molecular Biology–Genetics and Biotechnology
Supervisor: Assoc. Prof. Dr. Arzu KARABAY KORKMAZ
JUNE 2006
İSTANBUL TECHNICAL UNIVERSITY ���� INSTITUTE OF SCIENCE AND TECHNOLOGY
REGULATION OF MICROTUBULE SEVERING
M.Sc. Thesis by
Şirin KORULU, B.Sc.
(521031218)
Department: Advanced Technologies in Engineering
Programme: Molecular Biology–Genetics and Biotechnology
Supervisor: Assoc. Prof. Dr. Arzu KARABAY KORKMAZ
JUNE 2006
İSTANBUL TECHNICAL UNIVERSITY ���� INSTITUTE OF SCIENCE AND TECHNOLOGY
M.Sc. Thesis by
Şirin KORULU, B.Sc. (521031218)
Date of submission : 8 May 2006
Date of defence examination: 8 June 2006
Supervisor: Assoc.Prof.Dr.Arzu KARABAY KORKMAZ
Members of the Examining Committee: Assist.Prof.Dr.Eda TAHİR TURANLI (İ.T.Ü.)
Assoc.Prof.Dr. Hakan GÜRVİT (İ.Ü.)
JUNE 2006
REGULATION OF MICROTUBULE SEVERING
İSTANBUL TEKNİK ÜNİVERSİTESİ ���� FEN BİLİMLERİ ENSTİTÜSÜ
MİKROTÜBÜL KESİLMESİNİN REGÜLASYONU
YÜKSEK LİSANS TEZİ Şirin KORULU
(521031218)
HAZİRAN 2006
Tezin Enstitüye Verildiği Tarih : 8 Mayıs 2006
Tezin Savunulduğu Tarih : 8 Haziran 2006
Tez Danışmanı : Doç.Dr. Arzu KARABAY KOKMAZ
Diğer Jüri Üyeleri: Yar.Doç.Dr. Eda TAHİR TURANLI (İ.T.Ü.)
Doç.Dr. Hakan GÜRVİT (İ.Ü.)
ii
ACKNOWLEDGEMENTS
I would like to thank Associate Professor Arzu Karabay Korkmaz for invaluable guidance, advice, and also for her motivation and morale support at difficult times.
I would like to thank Ayşegüls ☺ (Ayşegül Yıldız and Ayşegül Dilsizoğlu) for being with me in all conditions. They are very enjoyable partners, as well as very good friends. It was easier to work in lab until late hours with such friends.
I would like to thank also Ceren Eke Koyuncu, Eyser Kılıç, Burcu Turanlı, Iraz Toprak Aydın for their friendship.
I would like to thank Prof. Peter W. Baas from Drexel University, Department of Neurobiology and Anatomy for providing me position in his lab. I would also like to thank Dr. Wenqian Yu for sharing all her technical experience during this lab work.
I would also like to acknowledge the funding agencies. This study was supported by TUBİTAK Career Project assigned for Dr. Arzu Karabay Korkmaz.
I would like to thank Ersin Koç for waiting for me ☺ and being the most enjoyable part of my life. He helped me to see that there is another life outside the lab ☺.
Finally, I would like to thank my family for their endless love and support. I always felt their courage.
3.1. Spastin Overexpression in Hippocampus Cells 46
3.2. Regulation of Spastin, MT Severing Protein, by Microtubule 50
Associated Proteins
4. DISCUSSION 55
5. CONCLUSION 60
REFERENCES 61
APPENDIX 67
RESUME 74
v
ABBREVIATIONS
AAA : ATPases Associated with various cellular Activities ATCC : The American Type Culture Collection BSA : Bovine Serum Albumine CNS : Central Nervous System DNA : Deoxyribonucleic Acid GFP : Green Fluorescent Protein HSP : Hereditary Spastic Paraplegia IR : Inter Repeats MAPs : Microtubule-associated proteins MTBR : Microtubule Binding Repeats MTOC : Microtubule Organizing Center MW : Molecular Weight NGF : Nerve Growth Factor PBS : Phosphate Buffered Saline RT : Room Temperature
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LIST OF TABLES
Page No
Table 2.1. List of plasmid constructs……………………………………... 19 Table 2.2. List of chemicals………………………………………………. 23 Table 2.3. List of kits……………………………………………………... 23 Table 2.4. List of equipments…………………………………………….. 23 Table 2.5. Fixative ingredients for hippocampus cells…………………… 33 Table 2.6. Constructs used for transfection………………………………. 39 Table 2.7. Fixative ingredients for RFL-6 cells…………………………... 40 Table 2.8. Dilution ratio for primary antibodies…………………………. 41 Table 2.9. Flowchart for primary antibody application…………………... 42 Table 2.10. Dilution ratio for secondary antibodies 42 Table 2.11. Flowchart for secondary antibody application for MAP1b
part…………………………………………………………….. 43
Table 2.12. Flowchart for secondary antibody application for MAP2c part……………………………………………………………..
43
Table A.1. Process measurements values of spastin overexpressing cells, day 4…………………………………………………………..
68
Table A.2. Process measurements values of p-60 katanin overexpressing cells, day 4…………………………………………………….
69
Table A.3. Process measurements values of GFP overexpressing control cells, day 4…………………………………………………….
69
Table A.4. Process measurements values of spastin overexpressing cells, day 6…………………………………………………………..
70
Table A.5. Process measurements values of p-60 katanin overexpressing cells, day 6…………………………………………………….
71
Table A.6. Process measurements values of GFP overexpressing control cells, day 6…………………………………………………….
72
Table A.7. Measurements values of MAP experiments……… 73
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LIST OF FIGURES
Page No
Figure 1.1 :MT assembly…………………………………………………. 1 Figure 1.2 :GTP sites in α- and β- tubulin subunits……………………… 2 Figure 1.3 :Dynamic instability…………………………………………... 3 Figure 1.4 :Model for MAP2 – MT interaction…………………………... 5 Figure 1.5 :Model for MAP1b gene organization………………………… 6 Figure 1.6 :Typical vertebrate neuron…………………………………….. 8 Figure 1.7 :Conformational change of AAA protein ring………………… 11 Figure 1.8 :Model for microtubule severing by katanin………………….. 12 Figure 1.9 :Domain localization of spastin……………………………….. 15 Figure 2.1 :Primary cell culture…………………………………………... 26 Figure 2.2 :Hippocampus dissection…………………….………………... 28 Figure 2.3 :Monolayer fibroblast cells…………………………………….. 36 Figure 3.1 :Spastin, p60, GFP overexpression in hippocampus cells
(day4)………………………………………………………….. 47
Figure 3.2 : Total process length per cell 48 Figure 3.3 :Spastin, p60, GFP overexpression in hippocampus cells
(day6)…………………………………………………….......... 49
Figure 3.4 :Average process numbers of cells…………………………...... 50 Figure 3.5 :MAP1b / Spastin co – expression…………………………….. 51 Figure 3.6 :MAP2c / Spastin co – expression…………………………….. 52 Figure 3.7 :MT mass change depending on the expressed protein ……….. 54 Figure 4.1 :Model for MAP–Microtubule Severing Protein Interaction…. 59
viii
LIST OF SYMBOLS
10 : Primary
20 : Secondary
H2O : Water Ab : Antibody dd : Double distilled fig. : Figure GFP : Green Fluorescent Protein g : Gram h : Hour IFs : Intermediate Filaments Lys : Lysine MAPs : Microtubule-associated proteins MFs : Microfilaments mRNA : Messenger-ribonucleic acid µl : Microliter µg : Microgram µm : Micrometer mg : Milligram ml : Milliliter MTs : Microtubules NF : Neurofilament PFA : Paraformaldehyde Tb : Tubulin
γγγγ-TuRC : γ-tubulin Ring Complex
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REGULATION OF MICROTUBULE SEVERING
SUMMARY
Microtubules are cytoskeletal polymers made of alpha and beta tubulin heterodimers. They are essential for the cell and have role in important cellular processes such as cell transport, cell motility and cell division. In neurons, MTs provide support for the growth and maintenance of the axonal and dendritic processes. They also serve as railroads along which organelles are transported within the axon.
Cells reconfigure their MTs by assembly and disassembly phases, known as dynamic instability. However, many cell types, such as neurons, have complex MT organization patterns, and it is difficult to explain their reconfiguration by dynamic instability, especially when the MTs are stabilized by some proteins. All these suggest that there are some other models for MT movement in the cell. According to our model, long MTs are stationary, but short MTs are mobile. Cells mobilize their MTs by severing them into short pieces. Severing activity is performed by enzymes such as katanin and spastin. Once short MTs elongate, they lose their mobility property.
Katanin and spastin are MT severing enzymes. Katanin is one of the best characterized MT severing proteins. It has two subunits, p60 and p80. P60 subunit has high homology with another MT severing protein, spastin. Nowadays, spastin is as popular as katanin because it is known that spastin mutation leads to neurological disorder, hereditary spastic paraplegia.
In this study, our aim was to have some further steps in characterization of spastin and also to identify the regulation mechanisms of MT severing by spastin and katanin. In this study, we specifically concentrated on MAP1b and MAP2c proteins that have protective role over MTs.
In the first part of the study, we have worked with hippocampus cells. In order to identify the role of spastin in neurons, we overexpressed GFP, p60 –katanin and spastin constructs in hippocampal cells. Cells were fixed at the particular time points following transfection, day2 and day4. After fixation, immunostaining was done and then by using primary and secondary antibodies cells were analyzed with fluorescent microscopy. Control cells, p60 –katanin overexpressing and spastin overexpressing cells were compared with each others.
In the second part of the study, MAP’s protective functions were analyzed in spastin and p60 –katanin overexpressing cells. P60 –katanin and spastin constructs were overexpressed with and without MAPs such as MAP1b and MAP2c in living fibroblast cells, RFL-6. Cells were fixed on the following day of transfection and stained with primary and secondary antibodies. After immunostaining, cells were analyzed with fluorescent microscopy.
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MİKROTÜBÜL KESİLMESİNİN REGÜLASYONU
ÖZET
Mikrotübüller, alfa ve beta tubulin heterodimerlerinden oluşan hücre iskeleti polimerleridirler. Hücre için son derece gerekli olup hücre taşınması, hücre hareketi, hücre bölünmesi gibi hücre için hayati önem taşıyan olaylarda görevlidirler. Sinir hücrelerinde, MTler hücrenin büyümesine destek sağlamakla beraber hücresel yapının şekillenmesinde, akson ve dendritlerin oluşumunda da etkilidirler. Ayrıca MTler organel gibi moleküllerin akson boyunca çift yönlü taşınmasında da demir yolu vazifesi görmektedirler.
Hücreler “dinamik kararsızlık” adı verilen mekanizma ile MTlerini yeniden yapılandırmaktadırlar. Ancak sinir hücrelerinde olduğu gibi, birçok hücre tipi karmaşık MT ağına sahiptir. Bu hücrelerin MT yapılanmalarını dinamik kararsızlık ile açıklamak, özellikle de MTler başka proteinler ile de etkileşim halindeyken oldukça güçtür. Bu bulgular MTlerin hücre içi hareketini açıklamaya çalışan başka modellerin de mevcut olduğunu düşündürmektedir. Üzerinde çalıştığımız modele göre (“kes ve koş” modeli), uzun MTler durağan, kısa MTler ise hareketlidirler. Bu modele göre, hücreler MTlerini hareketli hale getirmek için onların katanin, spastin gibi proteinler tarafından kesilip, küçük parçalara ayrılmasını sağlamaktadırlar. Kısa MTler tekrardan uzun hale geçtiklerinde ise hareket yeteneklerini kaybetmektedirler.
Katanin ve spastin MT kesici enzimlerdir. Katanin, karakterizasyonu en güzel yapılan MT kesen proteinlerinden biridir. İki alt üniteden oluşur, p60 ve p80. En az onun kadar popüler olan diğer bir MT bölme proteini de spastindir ve p60–katanin alt ünitesi ile büyük benzerlik göstermektedir. Spastinin en fazla ilgi çeken proteinlerden biri haline gelmesinin nedeni de bu proteinin MT temel hücre biyolojisini nörolojik hastalıklarla birleştirmesidir. Spastinin mutasyonunda kalıtsal spastik parapleji rahatsızlığına neden olduğu bilinmektedir.
Bizim bu çalışmadaki hedefimiz spastinin karakterizasyonunda yeni adımlar atabilmek, MTlerin spastin, katanin tarafından kesilmesinin mikrotübül ilişkili proteinler tarafından düzenlenmesini aydınlatmaya çalışmaktır. Çalışmanın bu kısmında MTler üzerinde koruyucu görevleri olan mikrotübül–ilişkili proteinler, özellikle de MAP1b ve MAP2c üzerinde durulmuştur.
Spastinin görevini aydınlatabilmek için hipokampüs hücrelerinde GFP, spastin, p60–katanin proteinlerinin ekspresyonu gerçekleştirildi. Hücreler transfeksiyonu takip eden 2. ve 4. günlerde sabitlendi. Birincil ve ikincil antikorlar ile boyandıktan sonra hücreler floresan mikroskobu ile incelendi. Kontrol hücreleri, katanin ve spastin proteinlerini aşırı eksprese eden hücreler aralarında karşılaştırıldı.
xi
Çalışmanın ikinci kısmında ise mikrotübül ilişkili proteinlerin, p60–katanin ve spastin eksprese eden hücreler üzerinde herhangi bir düzenleyici rolünün olup olmadığı aydınlatılmaya çalışıldı. P60–katanin ve spastin proteinleri mikrotübül ilişkili proteinlerin varlığında ya da bu proteinler olmadan fibroblast hücrelerinde eksprese edildiler. Transfeksiyonu takip eden günde hücreler sabitlendi. Birincil ve ikincil antikorlarla işaretlendikten sonra hücreler floresan mikroskobunda incelendiler.
1
1. INTRODUCTION
1.1. Cytoskeleton
In all eukaryotes, there are fibrous proteins in the cytosol. These proteins are
microfilaments, microtubules and intermediate filaments. They are collectively
called “cytoskeleton”. These cytoskeletal fibers give the cell strength and rigidity.
They also have control on movement within the cell, especially microtubules (MTs)
have very important role in cell division (Lodish, 1995).
1.1.1. Microtubules
Among the cytoskeletal proteins, microtubules are thought to have the most
important roles, especially in generation of cell shape and polarity, cell division, cell
growth and intracellular organelle transport.
Microtubules are polymers of α- and β- tubulin subunits. These subunits are
arranged in a cylindrical tube 24 nm in diameter. There are both lateral and
longitudinal interactions between the tubulin heterodimer subunits. These
interactions maintain the tubular form of microtubules (Lodish, 1995; Vale et al.,
1999).
Figure 1.1: MT Assembly
2
Each α-β tubulin heterodimer binds two molecules of GTP. One of the GTP-binding
sites is located on α-tubulin; second site for GTP-binding is located on β-tubulin.
GTP binding to the α-tubulin site is irreversible but on the β-tubulin site, GTP
binding is reversible; thus GTP can be hydrolyzed to GDP.
Figure 1.2: GTP sites in α- and β- tubulin subunits
In addition to α- and β- tubulins, there is a special third type of tubulin, γ-tubulin. It
is located in the centrosomal matrix. In animal cells, centrosomes are primary sites
for microtubule nucleation. Microtubules are thought to be nucleated from γ-tubulin
ring complexes (γ-TuRCs) and these microtubules nucleated from γ-TuRCs have
minus ends that are physically capped. These caps prevent minus-end polymerization
and depolymerization (Lodish, 1995; McNally et al., 2002).
1.1.1.1. Dynamic Instability of Microtubules
Microtubules continuously switch between growth and shrinkage phases. Growth
phase occurs by polymerization of tubulin at MTs ends while depolymerization
occurs by loss of tubulin subunits from their ends. This process is called “dynamic
instability”. Dynamic instability is characterized by the coexistence of polymerizing
and depolymerizing MTs. It is thought to be a function of GTP hydrolysis. GTP-
tubulin is incorporated at polymerizing MT ends, the bound GTP is hydrolyzed
during or soon after polymerization, and Pi is released. Thus, the MT lattice is
predominantly composed of GDP-tubulin (McNally et al., 1998; Hartman et al.,
1998).
3
Rapid loss of GDP-tubulin subunits and oligomers from the MT end is termed
depolymerization. Depolymerizing MTs can also transit back to the polymerization
phase, “rescue”. If the polymerizing MTs transit to the depolymerization phase, it is
called “catastrophe”. With the catastrophe, MTs switch to a rapid shortening phase
(Walker et al., 1988, 1991).
Figure 1.3: Dynamic instability
The dynamic behavior of microtubules is essential for fundamental processes in
eukaryotic cells such as cell division, cell differentiation, nerve growth, organizing
large intracellular compartments such as Golgi apparatus and endoplasmic reticulum
as well as for transporting small membrane carrier vesicles in the endocytotic and
secretory pathways. Also, during the mitotic cycle, microtubules are primarily
needed components of the mitotic spindle for proper segregation of chromosomes
and specifying the position of the cleavage furrow (McNally et al., 1993; Quarmby,
et al., 2000; David, et al., 1999).
There are also some other proteins that control the microtubule behaviors. They are
called microtubule-associated proteins (MAPs). They bind to microtubule walls and
promote microtubule polymerization. Other proteins such as OP18, XKCM1 increase
the frequency of catastrophes and thus, promote disassembly of microtubules from
MT ends (Hartman et al., 1998).
4
1.1.2. Microtubule Associated Proteins (MAPs)
Microtubule Associated Proteins (MAPs) control MT behaviors. It has been
proposed that MT – MAP interaction is predominantly electrostatic. MAPs are
positively charged and act by screening negative charges (highly acidic sites) on the
C – terminal domain of both in α- and β- tubulin. MAPs have N – terminal
projection domains, so that they can crosslink and bundle MTs. This promotes rescue
and causes stabilization. (Nogales, 2000; Desui et al., 1997).
Neuronal MAPs can be classically divided into two groups: (1) Very high molecular
weight polypeptides such as MAP1 and MAP2, which are abundant in adult brain,
(2) intermediate – sized proteins, such as tau. There is also MAP4 in non – neuronal
cells (Matus, 1988).
MAP2, MAP4 and tau have conserved C – terminal MT binding domain with three
or four pseudo repeats. Each repeat represents a MT binding site and is composed of
31 – 32 amino acids including several basic residues. MAP1 has only one N –
terminal MT binding domain which is acidic rather than basic (Nogales, 2000).
Most of the identified MAPs are thought to be regulated by phosphorylation.
Phosphorylation decreases MAPs ability to bind MTs by weakening the electrostatic
interaction between MTs and MAPs. When the phosphorylation increases, MAPs are
inhibited in MT stabilization ability.
1.1.2.1. MAP2 and MAP2c
MAP2 is a monomeric protein that has four isoforms 2a, 2b, 2c and 2d. MAP2 shares
homology in its MT binding domain with tau and MAP4. It stimulates the growth of
MTs in vitro by promoting nucleation and tubulin subunit addition at MT ends. MT –
MAP2 binding also leads to stabilization of MTs, in other terms reduction in their
dynamic instability and increase in MT rescue (Halpain et al., 2000).
Multiple isoforms of MAP2 are encoded by a single gene as a result of differential
alternative splicing mechanisms. MAP2a, MAP2b are high MW isoforms, but
MAP2c and MAP2d are low MW isoforms (Halpain et al., 2000). MAP2a and
MAP2b are mainly found in dendrites and cell body while MAP2c is particularly
5
pronounced in developing axons (Mandelkow et al., 1992). MAP2c is also
considered as juvenile MAP and it is expressed perinatally in rats, coincident with
the period of maximal dendritic outgrowth and synaptogenesis, then it is replaced by
MAP2a, MAP2b postnatally. Only some Central Nervous System (CNS) regions that
undergo neuritigenesis throughout postnatal life such as olfactory bulb, retina,
continue to express MAP2c at high levels into adulthood. This leads to the idea that
MAP2c has a specific function associated with dendritic outgrowth and
synaptogenesis (Mandelkow et al., 1992; Halpain et al., 2000).
Bloom and Valle suggested that MAP2 may be divided into two structural domains
(see fig. 1.4). C terminal domain has MT binding site which is positively charged. It
contains three 18 – residue MT binding repeats (MTBR) separated by 13 – 14
residue inter – repeats (IR). This domain promotes MT assembly. N terminal domain
is predominantly negatively charged and represents portion of MAP2 observed as a
projection of MT surface. Differently, the projection domain of MAP2 does not bind
to the MTs and is thought to extend into the solution, away from MT surface. This
projection domain contains binding sites for regulatory subunits of protein kinases
and these kinases have role in MAP2 regulation via phosphorylation (Vallee et al.,
1983; Mandelkow et al., 1992; Milligan et al., 2002).
Figure 1. 4: Model for MAP2 – MT interaction
6
1.1.2.2. MAP1b
MAP1b is one of the first MAPs to be expressed during embryonic development of
the nervous system. It is also known as MAP1x, MAP1.2 or MAP5. Molecular
weight of the protein is about 320kDa and its structure is filamentous with a small
spherical segment at one end (Kunkel, 1994; Taniquchi et al., 1997; Propst et al.,
2000). MAP1b is a multimeric protein complex that contains one heavy chain
(regulatory subunit) and at least one light chain (active subunit). There is a MT
binding domain in the N – terminal half of the heavy chain. This region is composed
of 21 times repeated highly basic KKE(E/I/V) motifs. It has been proposed that this
positively charged domain has an α – helical structure that binds to a negatively
charged α – helical domain at the C – terminus of β – tubulin which is on the outer
surface of the MT (Tögel, et al., 1998; Propst et al., 2000; Franzen et al., 2001).
Beside its MT binding ability, MAP1b can also bind to actin filaments; hence,
MAP1b is a link between these two proteins that form the growth cone cytoskeleton.
MAP1b is the earliest MAP expressed in the developing nervous system and is
abundant early in development. The level decreases in the adult, but it is still high in
adult dorsal root ganglion (DRG) neurons and sciatic nerve axons (Kunkel et al.,
1994; Fischer et al., 2000).
Figure 1. 5: Model for MAP1b gene organization
7
Localization studies have shown that MAP1b expression is much higher in actively
growing and developing neurons. It is also present in axons, cell bodies and dendrites
of neurons and in glial cells. Because MAP1b is especially expressed in axons during
their initial outgrowth, it has been suggested that MAP1b plays important role in
neurogenesis. When phosphorylated, it causes growth cone MTs to be in a
dynamically unstable form which is necessary for axonogenesis (Noble et al., 1989;
Kunkel et al., 1994; Tögel, et al., 1998; Bomont et al, 2003).
MAP1b is regulated by phosphorylation and dephosphorylation. Two modes were
identified for MAP1b phosphorylation. Mode I may be catalyzed by proline directed
protein kinases (PDPK), whereas the mode II is due to the action of casein kinase II
(CKII). MAP1b expression decreases after neuronal maturation; phosphorylation
state of MAP1b is also modified. Mode I phosphorylation disappears while the mode
II phosphorylation is still present in adult MAP1b (Avila et al., 1994).
1.2. Neuronal Cytoskeleton
Neurons develop from mitotic cells of ectodermal origin. After several divisions,
these cells begin to express neuron specific proteins. Human nervous system consists
of over 1011 neurons associated with over 1012 supporting glial cells. Neurons are non
– dividing cells, in order to transmit signals they stop dividing early in development
(Baas, 1999). Typical neuron has an enlarged cell body that contains a nucleus and
most of the cytoplasmic organelles. Cell body is also the place where all the neuronal
proteins are synthesized. Neuron has two different extensions from the cell body.
Branching ones are called dendrites and long one is called axon. Axons send
information over long distances, dendrites act as receptors for incoming information
(Baas, 2002). In humans, axons can grow to enormous length. This leads to
questions: “How does the neuron support and maintain such a long process?”, “How
can materials be transported along the axon?” The general answer lies under the
“neuronal cytoskeleton”. Neuronal cytoskeleton is composed three types of
filamentous proteins. Microtubules (MTs), microfilaments (MFs) and intermediate
filaments (IFs). Each has different types of subunits, tubulin for MTs, actin for MFs,
and a family of related proteins for IFs.
8
1.2.1. Neuronal Polarity
Neurons are the most polarized cells in nature. They contain one axon and multiple
dendrites. In axons, there are long MTs oriented with their plus ends away from the
cell body and MT polarity is uniform; whereas microtubules in the dendrites are
short and they have mixed polarity orientation (Baas, 1989; 1999; Vale et al., 1999,
Ahmad, et al., 1999). Another difference between axon and dendrites is
compartmentalization of some organelles. For example, dendrites have ribosomes
and Golgi elements, but axons do not. This explains why each process has
specialized roles during neuronal activities.
The most important question in neuronal polarity is that, how the neurons know to
extend a single axon and multiple dendrites? Broad agreement is that, this event is
due to changes in the cytoskeleton. Laboratory studies showed that when expression
of tau, major axonal MAP, was suppressed, transformation of immature neurite into
an axon was curtailed. This indicates that MT stability might be the basis of axonal
differentiation. However, the mechanism is not clear, since MAPs such as tau are
also involved in many activities, not just in MT stabilization (Baas, 2002).
Besides maintaining the cell shape, neuronal cytoskeleton is also important for
axonal transport. There is no protein synthesis machinery in axons and dendrites;
hence they can not synthesize tubulin subunits locally. Therefore, proteins and
tubulins must be transported in some form. There are two types of transport; fast and
slow transport.
Figure 1. 6: Typical vertebrate neuron
9
Fast transport is responsible for movement of organelles, and the travel rate is 100-
400 mm per day. Slow transport moves cytoskeletal proteins and soluble enzymes
and travel rate is 0.1 – 3 mm per day. This type of transport is especially important
for neuronal growth and process maintenance (Hirokawa, N., 1993; Baas, 1999).
1.3. Microtubule Severing
In many cell types, such as fibroblasts, minus-ends of microtubules are anchored
near the centrosome, whereas the plus-ends are oriented towards the cell periphery.
In other cells, such as epithelial cells and neurons, non-centrosomal microtubules are
needed for the activity of differentiated cell. There are three possible ways to
produce non-centrosomal microtubules: (1) the release of microtubules originally
anchored at the centrosome, (2) de novo nucleation and growth of microtubules in
the cytosol, (3) severing of microtubules at sites remote from the centrosome
(Quarmby, et al., 2000; Quarmby, et al., 1999). Recent studies support the idea that
microtubule severing is an important source of non-centrosomal microtubules. Most
of the newly formed minus-ends (as a result of microtubule severing) seem to be
stable (Quarmby, et al., 1999).
When it comes to possible roles of microtubule severing, this mechanism may play a
role in regulating poleward flux of tubulin in the metaphase spindle during cell
division, degradation of sperm axonemal microtubules after fertilization (in sea
urchin oocytes), microtubule reorganization during the transition from interphase to
mitosis in dividing cells, and the release of centrosome-nucleated microtubules
(Lohret et al., 1998).
Microtubule severing also plays a role in specific activities of differentiated cells. For
example in neurons, it is important in neuronal branching and axonal growth. The
effect of microtubule severing on neuronal cells was investigated by experiments that
inhibited the microtubule severing ATPase katanin activity. When anti-katanin
antibodies were injected into neurons, centrosomal microtubules accumulated and
the neuronal process did not occur (Ahmad, et al., 1999). According to the
experimental studies, the following model was developed for the effects of katanin
inhibition on process outgrowth. In control cells with active katanin, microtubules
10
are nucleated from centrosome and they are rapidly released by katanin after their
lengths become a few microns. Then, motor proteins transport microtubules outward
towards the cell periphery. These severing activities ensure that microtubules remain
relatively short. On the other hand, in experimental cells with inactivated katanin,
microtubules are not released from centrosomes. The number of individual
microtubules cannot be increased by severing the microtubules. As a result of all
these events, the process outgrowth is inhibited (Ahmad, et al., 1999). This supports
the idea that centrosomal katanin, so microtubule severing is important for the
production of non-centrosomal microtubules (Quarmby, et al., 2000).
As already mentioned, dendrites and axons contain large numbers of non-
centrosomal microtubules that are essential for architectural support and also act as
railway for the transport of materials along the axon (Dent, et al., 1999, Joshi, et al.,
1998). There are two possible mechanisms for the formation of non-centrosomal
microtubules in neuron cells: (1) these non-centrosomal microtubules are derived
from in situ nucleation and assembly, or (2) these non-centrosomal microtubules are
transported from the cytosol as polymers. As axons are incapable of locally
synthesizing the tubulin subunits, microtubules nucleated at centrosomes must be
actively transported from their sites of synthesis within the cell body of the neuron
down the axon in the form of assembled microtubule polymer (Baas, et al., 1997). If
the second mechanism occurs, these microtubules are probably produced by
microtubule severing (Quarmby, et al., 1999, Quarmby, et al., 2000).
1.3.1. AAA Family Proteins
Katanin and spastin are members of a large protein family, AAA which stands for
ATPases Associated with various cellular Activities. This family proteins play
important role in numbers of cellular activities including proteolysis, protein folding,
membrane trafficking, cytoskeletal regulation, organelle biogenesis, DNA replication
and intracellular motility (McNally et al., 1993; Vale et al., 2000).
The common feature of the AAA superfamily is an ATPase domain. This domain is
composed of about 220 amino acids. It is known that AAA domains assemble into
oligomeric structures and this allows proteins to change their shapes during ATPase
cycle. ATP binding induces structural rearrangements at the interface region of AAA
11
proteins. This increases interactions between adjacent AAA domains, also increases
interactions between AAA protein and its target (Vale et al., 2000; McNally, F. et al.,
2000).
Figure 1.7: Conformational change of AAA protein ring
Ring-like structures are useful for AAA enzyme mechanism. This allows subunits to
switch between tense and relaxed states in a concerted manner. These structures also
provide framework for binding target proteins at multiple sites. If ring-binding sites
change their positions, this will also cause tension application to bound protein (Vale
et al., 2000).
1.3.1.1. Katanin
Katanin is the most well characterized microtubule-severing protein. It was first
purified from sea urchin eggs. It is a heterodimer protein consisting of two subunits.
Enzymatic subunit is 60kD (p60) and it carries out the ATPase and severing
reactions. Other subunit is 80kD (p80) and it localizes katanin to the centrosome and
regulates microtubule-severing activity of p60 subunit (Vale et al., 1999; Quarmby,
et al., 2000). N-terminal domain of p60 subunit binds microtubules and C-terminal is
AAA domain (Quarmby, et al., 2000; McNally, K. et al., 2000).
AAA domain of p60 affects the binding affinity of the adjacent microtubule-binding
domain, and tight binding occurs in nucleotide states (ATP). This stabilizes p60 rings
(Vale et al., 1999). N-terminal of p80 subunit is composed of WD40 repeat (proline-
rich) domain and a C-terminal domain is required for dimerization with catalytic p60
subunit. Studies showed that WD40 repeat domain of p80 is required for spindle pole
localization of katanin. WD40 domain probably binds to another spindle pole protein
(McNally, K. et al., 2000). Although p60 shows its ATPase and severing activity in
the absence of p80 subunit, p80 cannot sever microtubules on its own.
12
Besides targeting katanin to the centrosomes it also enhances severing capacity of
p60. Association of the two subunits increases their affinity for microtubules and also
microtubule-severing activity (Hartman et al., 1998; Quarmby, et al., 2000; Ahmad
et al., 1999).
Katanin is a microtubule stimulated ATPase; thus microtubule concentration affects
the enzyme activity. Katanin forms ring structures and the ring formation occurs only
when katanin subunits bind to adjacent tubulin subunits in the microtubule wall
(McNally, F. et al., 2000). If microtubules are not present at the centrosome, katanin
is distributed in the cytoplasm. Once microtubules are nucleated at the centrosome
tubulin-katanin interactions make ring formation occur (Baas et al., 1997). At low
microtubule concentrations (<2µM), ATPase activity increases with increasing
microtubule concentration; but at higher microtubule concentrations, ATPase activity
decreases until it approaches basal levels.
This ATPase behavior of katanin is unusual, and there are some explanations for the
unusual ATPase behavior of katanin. Katanin binds microtubules at two sites; this
increases local microtubule concentration by cross-linking and thereby stimulates
katanin’s ATPase activity. At higher microtubule concentrations, the ratio of katanin
to microtubules is lower, less cross-linking occurs, thus less ATPase stimulation will
be observed. A second explanation is about katanin oligomerization into rings.
Microtubules promote p60-p60 oligomerization and oligomerization stimulates
ATPase activity. Low microtubule concentrations facilitate oligomerization because
p60 monomers are more likely to bind near one another on the microtubule. On the
other hand, when the microtubule concentration is high, this will inhibit p60
assembly by sequestering p60 monomers at non-contiguous sites (Vale et al., 1999;
Hartman et al., 1998).
Figure 1.8: Model for microtubule severing by katanin
13
The following question is “how does katanin sever microtubules?” Studies on
katanin gave rise to a model of microtubule severing. Katanin-ADP is monomeric
molecule. When katanin exchanges its ADP for ATP, p60-p60 affinity is enhanced,
and then oligomerization on microtubule is most efficient. Oligomerization allows
binding of multiple katanin subunits to multiple adjacent tubulin subunits in the
microtubule and 14-16 nanometer katanin ring is formed. Once a complete katanin
ring is assembled on the microtubule, the ATPase activity of katanin is stimulated.
As a result of ATPase reaction, phosphate group is released. Katanin undergoes a
conformational change. This creates a pulling or pushing force on the tubulin
subunits, leading to destabilization of tubulin-tubulin contacts. Katanin-ADP
monomer has lower affinity for other katanin molecules and for tubulin subunits.
This leads to the dissociation of complex and the recycling of the katanin (Vale et al.,
1999; Quarmby et al., 2000; McNally, F. et al., 2000).
Because of being hexamer, the katanin ring cannot be docked on the sides of
microtubule in such a way as to create identical interactions between katanin
subunits and tubulin subunits. There are two possible solutions for this paradox:
katanin hexamer can be docked inside the lumen of the microtubule. According to
this model, katanin monomers enter microtubule through dynamic lattice defects in
the microtubule wall and oligomerization occurs inside the microtubule. Second
solution is assembly of higher-order oligomers of hexameric rings on the outside of
the microtubules. Unlike a single hexameric ring, these can form multiple
homologous contacts with tubulin subunits in the microtubule wall. Coordinated
conformational changes in both structures lead to disrupt tubulin- tubulin interactions
(McNally, F. et al., 2000).
1.3.1.2. Spastin
Spastin is a member of AAA family and recently became protein of interest because
it merges the fundamental cell biology of MTs with a neurological disorder. Spastin
mutation leads to a genetically inherited disease spastic paraplegia.
14
There are also some other genes identified that are responsible for the disease. Most
frequent (~40%) form is due to SPG4 locus mutation which encodes spastin protein.
The mutations can be missense, nonsense, splice site mutation, deletion or insertion
in spastin gene (Errico et al, 2002).
Hereditary Spastic Paraplegia (HSP) can be in pure or complicated form. In pure
form weakness and spasticity of lower limbs are main characteristics. Patience
usually experience difficulties in walking. When the disease is in complicated form,
it has some additional neurological abnormalities such as retinopathy, deafness and
ataxia (Errico et al., 2002; Sherwood et al., 2004).
The degenerative process of the disease is interesting. It selectively affects some of
the longest axons in central nervous system. Corticospinal axon is most severely
affected. Next one is dorsal column. The degenerative process starts from distal ends
of these axons and proceeds toward the cell body. This is called “dying back”
axonopathy and the reason of such mechanism is still unclear (McDermott et al.,
2003).
There are hypothesis for the situation. Mutant spastin disrupts MT dynamics by
causing impairment of organelle transport on MT network. Supporting the
hypothesis, studies with rat cortical neurons showed that there was a decrease in
kinesin staining in mutant spastin overexpressing cells. Also in the same study, it was
observed that spastin do not localize in axonal and dendritic processes while mutant
spastin extended into the axons but not into dendrites (McDermott et al., 2003).
Another degeneration hypothesis is spastin can cause degeneration by diminishing
the supply of short MTs required for process generation. Mutant spastin lacks MT
severing activity, so that short MTs can not be formed via severing activity of spastin
(Baas et al, 2005).
Spastin is a member of AAA protein family. It belongs to the meiotic subgroup
which also contains proteins involved in vesicle trafficking and MT dynamics. P60 –
katanin is also a member of this group and it is the most characterized one. Spastin
shares great homology with p60 –katanin within AAA domain but they do not have
homology in their N – terminal region. Because of this homology, spastin is thought
to be MT severing protein as p60 –katanin. To test the hypothesis, several cell
15
culture experiments were done and overexpression of wild type spastin really caused
disassembly of MT cytoskeleton. Drosophila studies also showed that spastin
overexpression in muscle erases their MT networks consistent with the idea that
spastin is a MT severing protein. The same study also showed that Drosophila
spastin (Dspastin) has a positive role in maintaining the synapse by encouraging
growth through increasing dynamic instability of MTs. One more hypothesis raised
from this study: proteins that destabilize MTs should facilitate synaptic growth
(Sherwood et al., 2004; Roll – Mecck, 2005).
Spastin is encoded by SPG4 (SPAST) locus and it is composed of 17 exons
(Fonknechten, 2000). Spastin is 616 amino acid long and approximately 67.2 kDa. It
contains two leucine – zipper and coiled – coil dimerization motif (Charvin et. al.,
2003; Hazan et al., 1999).
Spastin is composed of three domains. N – terminal region contains putative
transmembrane region TM, MIT is a microtubule interacting and trafficking domain.
This domain is well conserved in spastin family. Final domain is ATP binding AAA
domain (Roll – Mecck, 2005).
Figure 1.9: Domain localization of spastin
Although having similarity with p60 –katanin in function, spastin needs lots of
further investigations. Even localization of spastin is controversial. It was reported
that there is a putative nuclear localization signal RGKKK at 7 – 11 positions of
human aminoacid sequence of spastin, but its sub cellular localization is still not
clear (Charvin et al., 2003).
Various studies were done by using different antibodies generated against the
protein. Some results pointed spastin to be a cytoplasmic protein however some
3276,6 4386,63 2326,69 4152,03 2785,79 4530,37 3435,17 2518,22 3701,27 TOTAL
73
Table A.7: Measurements values of MAP experiments
MAP2c/spastin MAP2c spastin GFP control MAP1b MAP1b/spastin
831,38 541,08 404,41 446,82 1720,97 522,62
533,89 861,1 381,39 425,67 1573,22 570,98
1004,84 953,15 387,02 450,55 1130,97 755,54
712,28 1134,73 433,71 509,86 1279,6 501,01
1007,58 1598,83 390,12 492,08 1934,62 566,83
851,68 1019,8 376,46 710,68 1290,62 359,56
916,14 1016,32 432,92 771,2 1355,34 487
844,19 1362,49 432,6 883,95 1616,11 511,74
1118,81 983,85 561,77 701,62
511,04
658,12 1409,53 468,24 801,09
642,47 1132,54 353,03
947,56 847,05 427,34
1129,43 1685,71 447,2
1167,97 821,93
1869,5
1223,62
1471,79
796,54
1156,65
1100,4
1363,89
1201,45
1167,97
1689,73
1038,95
1213,67
MAP2c/Hsp map2c spastin
Mean 1063,865 Mean 1097,722 Mean 422,7854
Standard Error 60,90478 Standard Error 85,10018 Standard Error 14,70377
Median 1069,675 Median 1018,06 Median 427,34
Standard Deviation 310,5546 Standard Deviation 318,4157
Standard Deviation 53,01519
GFP control MAP1b MAP1b+Hsp
Mean 619,352 Mean 1487,681 Mean 531,8133
Standard Error 54,27996 Standard Error 94,95081 Standard Error 34,63322
Median 605,74 Median 1464,28 Median 511,74
Standard Deviation 171,6483 Standard Deviation 268,5615
Standard Deviation 103,8996
74
RESUME
Şirin Korulu was born in Bulgaria in 1980. After getting her high school diploma from Bursa Boys High School in 1999, she has continued her undergraduate degree at Istanbul Technical University, Department of Molecular Biology and Genetics in 1999. She had her Bachelor degree in 2003. She has continued to her graduate studies at Advanced Technologies in Molecular Biology – Genetics and Biotechnology program in the Istanbul Technical University. She has been also working as a research assistant in Department of Molecular Biology and Genetics. She is still pursuing her studies in the same department.